A METHOD FOR OBTAINING AXENIC ALGAL CULTURES USING THE ANTIBIOTIC CEFOTAXIME WITH EMPHASIS ON CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA)1

Axenic cultures of the tropical green seaweed Cladophoropsis membranacea (C. Agardh) Boergesen were obtained by cutting 2-mm apical tips from fast-growing unialgal filaments and incubating them in 1/2PES containing 100 μg · mL^?1 cefotaxime. After 1 week, apical tips were cut from the newly grown plantlets, washed through a series of sterile drops of seawater, and incubated in sterile 1/2PES. Plantlets were screened for the presence of bacteria by incubating droplets of the culture medium on peptone agar plates and by examining DAPI-stained filaments. Ninety to one hundred percent of the plantlets obtained with this treatment were axenic. Cefotaxime was also effective against cyanobacteria but of only limited value in rhodophytes.     

芬芳桔色藻的一个新变种

芬芳桔色藻的一个新变种梁良弼毕列爵（福建师范大学生物系福州３５０００７）（湖北大学生命科学系武汉４３００６２）关键词芬芳桔色藻,芬芳桔色藻小型变种,云南ＡＮＥＷＶＡＲＩＥＴＹＯＦＴＲＥＮＴＥＰＯＨＬＩＡＯＤＯＲＡＴＡ（ＷＩＧＧＥＲＳ）ＷＩＴＴＲＯＣＫ...     

EFFECTS OF DESICCATION AND ILLUMINATION ON PHOTOSYNTHESIS AND PIGMENTATION OF AN EDAPHIC POPULATION OF TRENTEPOHLIA ODORATA (CHLOROPHYTA)1

The photosynthetic rates of Trentepohlia odorata (L.) Martius growing on wall surfaces in Singapore changed throughout the day with a maximum in midmorning and decreasing thereafter during the day. Optimum temperature for photosynthesis was 25° C. Different levels of air humidity also affected photosynthetic rates with low relative humidity reducing the rates and efficiency of photosynthesis. Our results suggested that T. odorata was able to maximize its rate of photosynthesis before photoinhibitory light levels were reached and that its growth might be dependent on high levels of atmospheric relative humidity, which may serve as a source of water supply for the alga.     

AXENIC CULTURES OF ACETABULARIA (CHLOROPHYTA): A DECONTAMINATION PROTOCOL WITH POTENTIAL APPLICATION TO OTHER ALGAE1

We developed an easy, reliable decontamination protocol for caps of Acetabularia acetabulum (L.) Silva; with minimal labor hundreds of caps can be decontaminated. In addition, cysts isolated from these caps do not exhibit dormancy and can be used immediately to establish large populations of axenic cell cultures. This method consists of three successive incubations: 1) proteinase K/sodium dodecyl sulfate/1,3-bis[tris(hydroxymethyl)-methylamino]propane for 1 h, 2) mild silver protein for 5 min, and 3) an antibiotic solution (neomycin, chloramphenicol, nystatin, ampicillin, streptomycin)for 3 days. This protocol eliminates bacterial, algal, fungal, and yeast contaminants. It is useful for decontaminating caps from lab cultures and caps collected from the wild and may also be effective in decontaminating the reproductive structures of other algae. Cyst dormancy was reduced from 15 weeks to less than 1 week, which represents a 40% reduction of the life cycle of A. acetabulum. We routinely obtained 90-100% gamete release from cysts 3-14 days after they were made axenic. The component of our culturing methods that allows gamete release without a “dormant” period is unknown.     

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram-positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter-sterilized supernatants of culture media of MG-active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.     

PRODUCTION OF AXENIC CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE) USING ANTIBIOTIC 1

Bacteria-free cultures of the prasinophyte Micromonas pusilla (Butcher) Monton and Parke, UTEX LB 991, were produced by intially determining the effects of several antibiotics on the growth of this alga and then using a combination of these antibiotics to eliminate associated bacteria, Micrononas pusilla was resistant ot penicillin G, neomycin, gentamicin and streptomycin at bactericidal concentrations but sensitive to chloramphenicol and polymixin B. Passage of M. pusilla through the sequence of antibioties penicillin G → neomycin → gentamician → kanamycin resulted in an axenic culture of M, pusilla this method should be suitable for producing axenic culture of other strains of M. pusilla.     

POLYMORPHIC BEHAVIOR OF ULVA LACTUCA (CHLOROPHYTA) IN AXENIC CULTURE. I. OCCURRENCE OF ENTEROMORPHA-LIKE PLANTS IN HAPLOID CLONES1

An isolate of Ulva lactuca L. was brought into axenic culture in both defined synthetic medium and enriched seawater. Haploid clones were established and followed through several generations in both media. Plants possessing distromatic, partially distromatic or completely tubular blades in cross sections, as well as individuals that were completely distromatic in one area of the blade and tubular in another all developed from the same swarmer population. Variations in basal area morphology also occurred. Progeny from swarmers of any of these morphological types showed a similar mixture of morphological variation, indicating non-mutational variability in phenotypic expression of the blade and basal areas. A mechanism by which such variation may occur without mutation is discussed.     

AXENIC TISSUE CULTURES FROM THE SPOROPHYTES OF LAMINARIA DIGITATA AND LAMINARIA HYPERBOREA (PHAEOPHYTA)

Starting material for the tissue cultures was the meristematic basal zone of the blade. Pieces treated 30–60 sec in hypochlorite solution were rinsed and placed on agar plates made from the artificial seawater ASP6 F2 solidified with 6 g agar l^?1. After 6 weeks colorless callus-like tissue grew out from some pieces. Treatment with activated charcoal removed some inhibiting substances from the agar medium as numbers of callus developing pieces increased on such plates. A combination of 10^?5 M NAA and 5 · 10^?7 M kinetin gave a yellow-brown tissue. A differentiation in the tissue from L. hyperborea was observed as well as the formation of meiospores, which grew out into male and female plants. Thalli of sporophytes were observed but they never reached a length of more than one mm before they died or changed to an irregular pattern of growth.     

NOTE ESTABLISHMENT OF AXENIC CULTURES OF ANABAENA FLOS-AQUAE AND APHANOTHECE NIDULANS (CYANOBACTERIA) BY LYSOZYME TREATMENT

A new method using lysozyme for the production of axenic cultures of Anabaena flos-aquae De Brebisson and Aphanothece nidulans P. Richter was developed. Cyanobacterial growth was not inhibited at concentrations up to 1.2 g·L⁻¹ of lysozyme, whereas the growth of heterotrophic bacteria was suppressed. At concentrations up to 0.8 g·L⁻¹ of lysozyme, ampicillin caused a reduction of heterotrophic bacteria. The axenic cultures of these strains were acquired through a simple treatment using 1.0 g·L⁻¹ of lysozyme without ampicillin. These cyanobacteria resisted digestion by lysozyme at our experimental concentrations, whereas bacteria were digested selectively. This method of purification seems to be especially useful with cyanobacterial species that are sensitive to antibiotics or other germicidal agents.     

A NOVEL TECHNIQUE FOR PREPARATION OF AXENIC CULTURES OF SYMBIODINIUM (PYRROPHYTA) THROUGH SELECTIVE DIGESTION BY AMOEBAE1

The marine amoeba Trichosphaerium Am-I-7 was used as a tool for preparing unialgal axenic cultures of nondigestible Symbiodinium and Porphyridium species. The resistance of these unicellular algae to the amoebal digestive enzymes, and the differential digestion of bacteria, protozoans, and other algae, resulted in cleansed cells of Symbiodinium and Porphyridium that remained in the amoebal food vacuoles. During multiple fission, the amoeba evacuated its food vacuoles and released the trapped and intact algae, which were then successfully cultured. This method of cleaning was especially useful with algal species that were sensitive to antibiotics or other germicidal agents.     

MITOSIS AND CYTOKINESIS IN SESSILE SPORANGIUM OF TRENTEPOHLIA AUREA (CHLOROPHYCEAE)1

An ultrastructure study of mitosis and cytokinesis in the sessile sporangium of Trentepohlia aurea (L.) Mart, was made to clarify the phylogenetic position of the alga. Mitosis was closed and centric at late anphase with cytokinesis involving the production of cleavage membranes by dictyasames between the numerous, well-separated daughter nuclei. Neither phycoplast nor phragmoplast microtubules were observed during cytokinesis. The lack of phycoplast microtubules and the presence of multilayered structures in flagellated cells suggest Trentepohlia is phylogenetically related to those green algae thought to have given rise to the land plants. The primitive type of mitosis and the lack of microbodies suggest that the ancestors of Trentepohlia may have branched off from this line relatively early.     

INHIBITION OF GROWTH OF ULVA EXPANSA (CHLOROPHYTA) BY ULTRAVIOLET-B RADIATION1

We examined the effect of ultraviolet-B radiation (UV-B, 290–320 nm) on the growth rate of the intertidal marine alga Ulva expansa (Setch.) S. & G. (Chlorophyta). Segments of thallus collected from a natural population were grown in outdoor seawater tanks. Combinations of UV-B-opaque screens, UV-B-transparent screens, and UV-B lamps were used to investigate the effects of solar UV-B and solar plus supplemental UV-B on the growth of these segments. Growth was measured by changes in segment surface area, damp weight, and dry weight. Growth rates of segments were inhibited under both solar UV-B and solar plus supplemental UV-B treatments. Growth rates were also inhibited by high levels of photosynthetically active radiation, independent of UV-B fluence. These results indicate that increases in UV-B resulting from further ozone depletion will have a negative impact on the growth of this alga.     

MORPHOGENESIS OF MONOSTROMA OXYSPERMUM (KÜTZ.) DOTY (CHLOROPHYCEAE) IN AXENIC CULTURE,ESPECIALLY IN BIALGAL CULTURE1

The typical morphology of Monostroma oxyspermum (Kütz.) Doty is lost in axenic culture. In synthetic media of the ASP type, it grows as a colony-like mass composed of round cells with numerous rhizoids. Such a mass is a fragile structure which falls apart upon shaking, or slight touch, into small cell-groups and single cells or cells with a long rhizoid. Only temporary saccate or monostromatic fronds appear and reach 1–2 mm in length when grown in enriched seawater media, but disintegrate and become a colony-like mass. The typical morphology is easily restored by adding at specific intervals filtrates of bacterial cultures and supernatant medium from axenic brown and red algal cultures to the basal medium (ASP7), or by reinfecting the Monostroma with an appropriate bacterial flora. Furthermore, the typical morphology in also maintained by bialgal cultures between Monostroma and other axenic strains of various species of seaweeds except the species belonging to the Chlorophyceae. Monostroma thus appears to utilize some substances released by most species of brown and red algae for its typical growth. Active substances released by bacteria, brown and red algae have not yet been identified and purified. However, it is demonstrated that in axenic cultures many species of seaweeds produce active extracellular substances which play an important role in growth and Morphogenesis of other species of seaweeds.     

EFFECT OF DISSOLVED OXYGEN PARTIAL PRESSURE ON THE ACCUMULATION OF ASTAXANTHIN IN CHEMOSTAT CULTURES OF HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)

The effect of dissolved oxygen partial pressure on the accumulation of astaxanthin in the green alga Haematococcus lacustris ( Gir.) Rostaf (UTEX16) was studied in N-limited continuous chemostat cultures. The steady-state astaxanthin content measured against culture volume, cell number, and biomass dry weigh of Haematococcus cultures was proportional to the dissolved O₂ partial pressure in the culture medium, over the range of 0–50% O₂ The steady-state biomass dry weight concentrations remained at between 0.52 and 0.57 g. L^-1 over the range of dissolved O₂ partial pressure studied. Steady-state cell densities at dissolved O₂ partial pressures above the air saturation level (1.13–1.58 × 10⁵ cells.mL^-1) were about half of that measured at lower dissolved O₂ partial pressures (2.42–2.63 × 10⁵ cells.mL^-1). Both biflagellated zoospores and nonmotile aplanospores were found at steady state. The fraction of nonmotile cells was higher at dissolved O₂ partial pressures above the air saturation level (94.44–98.01%) than at dissolved O₂ partial pressure below the air level (79.64–86.12 and 91.75% ).     

AXENIC CULTIVATION OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA SHUMWAYAE AND OBSERVATIONS ON FEEDING BEHAVIOR1

Pfiesteria shumwayae Glasgow et J. M. Burkh. [=Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester] is a heterotrophic dinoflagellate commonly found in temperate, estuarine waters. P. shumwayae can feed on other protists, fish, and invertebrates, but research on the biochemical requirements of this species has been restricted by the lack of axenic cultures. An undefined, biphasic culture medium was formulated that supported the axenic growth of two of three strains of P. shumwayae. The medium contained chicken egg yolk as a major component. Successful growth depended on the method used to sterilize the medium, and maximum cell yields (10⁴ · mL^?1) were similar to those attained in previous research when P. shumwayae was cultured with living fish or microalgae. Additionally, P. shumwayae flagellate cells ingested particles present in the biphasic medium, allowing detailed observations of feeding behavior. This research is an initial step toward a chemically defined axenic culture medium and determination of P. shumwayae metabolic requirements.     

STRUCTURE OF THE ANISOGAMETES OF THE GREEN SIPHON PSEUDOBRYOPSIS SP. (CHLOROPHYTA)1

The fine structure of the male and female gametes of Pseudobryopsis, particularly that of the flagellar apparatus, is compared with that of swarmers of other green algae. There is general similarity, with differences in detail, to the Ulvales and other green siphons that have been studied. The similarities include overlapping basal bodies, the capping plate type of connective between basal bodies, terminal caps, and system II fibrous roots (rhizoplasts). The capping plate of the female gamete differs from that in other green siphons and the Ulvales in form and in the presence of a faint striation. A diagram illustrating the actual arrangement of the components of the flagellar apparatus is given, along with a discussion of the fact that the mirror image of the true arrangement has been given in some reports on ulvaphycean algae.     

AXENIC TISSUE CULTURE AND MORPHOGENESIS IN PORPHYRA YEZOENSIS (BANGIALES, RHODOPHYTA)

To better understand developmental phenomena in macroalgal tissue culture, we examined the morphogenesis of Porphyra yezoensis Ueda (strain TU-1) cultured aseptically in defined synthetic media . Generally, the filamentous thalli (sporophyte; conchocelis phase) of P. yezoensis were densely tufted with uniseriate filaments. The foliose thalli (gametophyte) were monolayered. In this study, axenic filamentous thalli retained their characteristic morphogenesis; there were no obvious differences between morphogenetic traits in unialgal and axenic conditions. However, conchospores, which might have developed into the foliose form under unialgal conditions, germinated into calluslike masses under axenic conditions. Most of the gametophytes gradually lost their typical morphogenesis after the first longitudinal cell division. Some of the calluslike masses developed rhizoidlike structures in several places or along the entire mass. Therefore, we concluded that P. yezoensis, in axenic cultures, loses its typical morphogenesis only during the gametophytic phase. The axenic tissue culture of Porphyra established in this study is a promising assay system for the identification of growth and morphogenetic factors.     

DIVERSITY,LIFE HISTORY,AND ECOLOGY OF TRENTEPOHLIA AND PRINTZINA (TRENTEPOHLIALES,CHLOROPHYTA) IN URBAN HABITATS IN WESTERN IRELAND 1

On the basis of field and culture investigations, five species of the genera Trentepohlia and Printzina were found to occur in urban habitats in western Ireland: Trentepohlia abietina (Flotow) Hansgirg, T. aurea (Linnaeus) Martius, T. iolithus (Linnaeus) Wallroth, T. cf. umbrina (Kützing) Bornet, and Printzina lagenifera (Hildebrandt) Thompson et Wujek. These species formed perennial populations on a variety of substrata. T. abietina occurred on bark of trees; T. cf. umbrina occurred on stone walls; and P. lagenifera grew on several substrata, mainly cement and asbestos sheeting. T. aurea and T. iolithus were found on old concrete and cement walls; in particular, the latter species formed characteristic, extensive, deep‐red patches on many buildings. In culture, best growth and reproduction of these species were observed at 10 and 15° C, 16:8 h light:dark. Both in culture and in the field, reproduction took place by release of biflagellate swarmers behaving as asexual spores, germinating to produce new plants without any evidence of sexual fusion; release of biflagellate swarmers in the field was generally observed in all seasons throughout a whole annual cycle. Confirmation of the occurrence of sexual reproduction in Trentepohlia was not obtained.     

IDENTIFICATION OF A MYOSIN-LIKE PROTEIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

A myosin-like protein was identified in vegetative cells of the unicellular green alga Chlamydomonas reinhardtii Dangeard. Polyclonal antibodies affinity purified against the heavy chain of slime-mold myosin recognized a 180,000 M_r protein in western blots of total protein extracts from three different strains, including cyt-1, a cytokinesis-defective mutant. Immunoblots of isolated chloroplasts indicated that some of the cellular myosin fractionated with chloroplasts, whereas tubulin did not. Evidence for the presence of at least one myosin gene was obtained by probing Southern blots of genomic DNA with a myosin heavy-chain gene fragment isolated from the green alga Ernodesmis verticillata (Kützing) Børgesen. Collectively, the immunological and molecular data identify at least one myosin heavy-chain gene and a myosin-like protein in vegetative cells of the model organism Chlamydomonas.